Ayşe DİNÇER, Burcu OKUTUCU, Figen ZİHNİOĞLU, Azmi TELEFONCU

Ayşe DİNÇER, Burcu OKUTUCU, Figen ZİHNİOĞLU, Azmi TELEFONCU

The use of immobilized enzymes in industry and medicine requires the development of new methods for immobilization. The increasing use of immobilized enzymes as catalysts in various processes is mainly due to their reusability. Furthermore, immobilization affords easy recovery of the enzyme from the substrates and products, and may result in enhanced enzyme stability and alters kinetic characteristics. Currently, enzyme therapy and oral controlled delivery of protein drugs are extensively studied by means of various immobilization techniques. For this is aim some natural polymers especially polysaccharides have been widely used because of their unique advantages, such as; non-toxic, biocompatible, biodegradable and abundant properties. Entrapment in ionotropic gels is one of the simplest methods of immobilization that provides immobilization under mild conditions and therefore results in minimum denaturation of the catalyst during the process.

-Glucosidases (E.C; 3.2.1.20) catalyze not only the hydrolysis of an -glucosidic linkage, but also the transglucosylation of an -glucosyl residue to various glucosyl co-substrates resulting the synthesis of new oligosaccharides, besides digestion, lysozomal catabolism of glycoconjugates and glycoprotein synthesis. Polygalacturonic acid(PGA) is a major component of naturally occurring water-soluble polysaccharide; pectin. Chitosan is a cationic polysaccharide, which consist of N-acetylglucosamine and glucosamine residues made from alkaline N-deacetylation of chitin.

In this study, baker’s yeast -glucosidase entrapped in PGA(2.5%) beads prepared by using gelation of CaCl₂(2%) with PGA, were coated with chitosan(0.2%). Immobilization yield and dimentions of spherical beads were determined by means of general procedures. Furthermore, optimum pH and temperature, kinetic constants(Km, Vmax), reusage, thermal, storage and pH stability of the -glucosidase beads were investigated in comparison with the free enzyme. Data showed that stability of the enzyme enhances by immobilization. Furthermore, in vitro release studies in various physiological pH’s were also investigated.

Cu/Zn SOD IN BRAIN CORTEX OF RATS EXPOSED TO ACUTE AND CHRONIC STRESS

Dragana Filipovic, Vesna Stojiljkovic, Jelena Kasapovic, Snezana Pejic, Snezana B. Pajovic, and Marija B. Radojcic

Laboratory of Molecular Biology and Endocrinology, Institute of Nuclear Sciences”Vinca”

11000 Belgrade, Serbia and Montenegro, email: dragana@rt270.vin.bg.ac.yu

The role of enzymic antioxidant defence system in reparation of cell injury induced by high concentration of reactive oxygen species (ROS) generated during different stress conditions is of great importance. We examined the changes in the concentration of Cu/Zn superoxid dismutase (Cu/Zn SOD) in the brain cortex cytosol of two months old male Wistar rats. The animals were exposed for 2h to either cold (4⁰C) or immobilization, as acute stresses, or for 21day to either isolation (LTI) or crowding (LTC) as chronic stresses. The LTI or LTC were also followed by the acute stresses. The concentration of Cu/Zn SOD was determined in the tissue cytosol fraction by Western immunoblotting and quantitative analysis on Gel Doc 1000.

The Western blot analysis of Cu/Zn SOD by the polyclonal antibodies, showed pronounced increase in the enzyme concentration in both acute stresses. The increase in Cu/Zn SOD was cca. 39% (p0.01) after 2h immobilization and cca 35% (p0.01) after 2h of cold-exposure. After the LTI and LTC, the concentration of Cu/Zn SOD in the brain cortex increased for cca. 25% vs. 12% (p0.05). The subsequent exposure of animals to acute immobilization did not affect the concentration of Cu/Zn SOD in LTI animals, but the enzyme increased for 4% (p0.01) in LTC animals. The changes in Cu/Zn SOD concentration were most pronounced after cold exposure and increased for additional 75% (p0.01) in LTI animals, and for 18% ( p0.05) in LTC animals.

The results of our measurements showed that both acute stresses lead to the significant increase of Cu/Zn SOD concentration. Also, both chronic stresses: LTI and LTC resulted in similar, but smaller increase in Cu/Zn SOD compared to the acute stresses. The additional 2h cold exposure was more potent acute stressor than immobilization, and caused the highest increase in Cu/Zn SOD concentration.

H.Güneş ÖZHAN^*, Çağhan Kızıl^*, Güvenç Görgülü^**, Gökhan SADİ^**, Feride Severcan^*** and Tülin Güray^**

Melatonin (5-methoxy-N-acetyltryptamine) is a pineal gland hormone that takes role in phase control of internal clock, core body temperature, development and ageing. Melatonin has received growing attantion in recent years due to its putative roles in preventing free-radical-induced oxidative damage. Melatonin has implications for disease processes, for instance neurodegenerative and cardiovascular diseases, which involve free radicals. Free radicals are also known to be responsible in aging.

Eight female Sprague-Dawley rats, three controls and three melatonin-treated, were employed. Effects of melatonin on activities of three antioxidant enzymes, namely Superoxide Dismutase (SOD), Catalase (CAT) and Glutathione Peroxidase (GPx), have been investigated from rat liver tissue.Furthermore, impact of melatonin on microsomal lipid peroxidation rate was also determined by TBA-RS test in order to acquire a better understanding of the role of melatonin as a free-radical scavenger and effects on microsomal membranes.

We observed significant increases in CAT (p < 0,05), GPx (p < 0,05) and SOD (p < 0,005) activities for melatonin treated samples. In addition, a considerable decrease in lipid peroxidation rate ( p < 0,05) was obtained for the treated samples which was compared with the findings obtained from FTIR specta.

Keywords: Melatonin, SOD, CAT, GPx, Lipid peroxidation, FTIR, antioxidant enzymes

P125

PREPARATION AND COMPARISON OF ALCOHOL BIOSENSORS BASED ON ALCOHOL OXIDASE IMMOBILIZED IN DIFFERENT IMMOBILIZATION MATERIALS

Erol AKYILMAZ, and Erhan DİNÇKAYA

For the determination of alcohol two biosensors based on alcohol oxidase immobilized in gelatin-alginate and gelatin--carrageenan were developed.

Alcohol oxidase (EC 1.1.3.13) catalysis the oxidation of primer aliphatic alcohols to aldehyde and hydrogen peroxide in the presence of oxygen which acts as a co-substrat for the enzyme.

Commercially available alcohol oxidase from C.Boidinii was immobilized on a dissolved oxygen probe, covered with an oxygen sensitive teflon membrane, by using gelatin-alginate and gelatin---carrageenan as the immobilization material in the presence of a croos-linking agent, glutaraldehyde. In the experiments a YSI Model 57 oxygenmeter was used. Measurements were carried out at 35⁰C with various alcohol concentrations under steady-state conditions. By using the biosensors developed the amount of oxygen consumed being proportionate to alcohol concentration was determined according th the reaction given below;

After obtaining the linear detection limits of the biosensors some optimization and characterization studies of the biosensors were done. For this purpose some parameters such as optimum pH, temperature, substrate specificity, thermal and storage stability were investigated. In the reproducibility experiments for 0.5 mM concentration of alcohol (n=10) the standard deviation and variation coefficient were found as  0.00632 and 1.28 % for the gelatin-alginate based alcohol biosensor. For the gelatin---carrageenan based biosensor the standard deviation and variation coefficient for 0.1 mM concentration of alcohol (n=10) were found as  0.00290 and 2.98 %, respectively.

Sezer OKAY¹, Evrim IPEK¹, Hulya ZEYTINOGLU¹, Mine KURKCUOGLU²

In this study, the mutagenic and antimutagenic effects of the essential oil of Origanum onites L. and carvacrol that are used in medicine, flavouring of food and crop protection were investigated. The mutagenic and antimutagenic activities were screened using Salmonella typhimurium strains TA98 and TA100, with or without S9 metabolic activation. No mutagenicity was found in the oil to the both strains either with or without S9 mixture. The oil and its major constituent carvacrol were finally tested for their antimutagenic activity with 30 min standard preincubation time. It was shown that both of them strongly inhibited mutagenicity induced by 4-nitro-o-phenylenediamine and 2-aminofluorene in both strains with or without S9, respectively. These results indicate significant antimutagenicity of the essential oil and carvacrol in vitro, suggesting its pharmacological importance for the prevention of cancer.

In the last decade, in addition to the traditional and acknowledged risk factors such as hypertension, smoking, gender, hypercholesterolemia, a number of biochemical compounds have been recognized as new risk factors of coronary artery disease (CAD). Homocysteine (Hcy), one of these compounds, has been revealed to be an independent risk factor for CAD. Methylenetetrahydrofolate reductase (MTHFR) is one of the three key enzymes in Hcy metabolism. The atherosclerotic potential on the mutation (677T) of MTHFR gene remains controversial. However, polymorphisms in coagulation factors, such as Factor V Leiden (FVL) have been established as a risk factor for arterial thrombotic diseases but their effects are still not clear for CAD.

We aimed to study these mutations in patients with CAD and normal controls. The case-control study included 117 patients with the diagnosis of CAD and 104 controls. The DNA was extracted from whole blood by Poncz method; we used Light Cycler and Real-Time PCR for mutations analyses. Although the prevalence of prothrombin 20210A and FVL was higher in CAD patients than control subjects, the difference was not statistically significance. Our data suggests that there may be an association between the MTHFR 677T gene mutation and the presence of CAD (p<0.05). We observed that the lipid profiles (LDL Cholesterol, Total Cholesterol and Triglyceride) were significantly increased in CAD patients, although the HDL Cholesterol was not found to be significant.

P128

INHIBITION OF MYELOPEROXIDASE BY TIAZOFURIN

Tatjana MOMIĆ, Vesna VASIĆ

Myeloperoxidase (MPO) is protein that exist in granulocytes and catalyse the conversion of H₂O₂ and chlorine into HOCl. To help clarify the role of this enzyme in bacterial cilling and inflammation, a protein inhibitor needs to be identified. The aim of this study was to investigate wheter tiazofurin, effective oncolytic agent in chronic granulocytic leukemia, has a inhibitory effect on MPO activity and to evaluate some properties of this inhibition. The inhibitory effect of tiazofurin on MPO activity was studied in human granulocytes. MPO activity was measured spectrophotometrically throught the oxidation of a syntetic substrate o-dianisidine in the presence of H₂O₂. Tiazofurin inhibited MPO activity in a dose-dependent but not time-dependent manner with an IC₅₀ value of 5x10^-2 M. Using this tiazofurin concentration, the inhibitory effect was monitored at different substrate concentrations. The highest granulocytes MPO activity was obtained with 0.20x10^-3 M H₂O₂. Concentrations of substrate higher then this value inhibited the enzyme activity. Km values obtained for control (sample without inhibitor) and samples with 5x10^-4 M, 1x10^-2 M, 5x10^-2 M tiazofurin were 0.12 mM, 0.098 mM, 0.086 mM and 0.050 mM, respectively. Since our results showed that increasing inhibitor concentration decreased both, Km and Vmax values, tiazofurine is a noncompetitive inhibitor for human granulocyte MPO.

MOLECULAR PATHOLOGY OF CYP1B1 GENE (CYT P4501B1) IN TURKISH PATIENTS

Sefayet BAGİYEVA¹, Rıza Köksal ÖZGÜL¹, Sinan M. SARICAOĞLU ², Cihan ÖNER¹ and Ay ÖĞÜŞ¹

1Hacettepe University, Department of Molecular Biology, Beytepe-Ankara,Turkey

Numune Research and Training Hospital, Department of Ophthalmology, Ankara,Turkey

sefaet@hacettepe.edu.tr

Primary Congenital Glaucoma (PCG) or Buphthalmos (GLC3) is an autosomal recessive disorder, associated with unknown developmental defect(s) in anterior chamber and manifests itself in early childhood, usually within the first year of life. Responsible gene for PCG phenotype is CYP1B1, the only known member of cytochrome P450 I subfamily of CYP1B. This gene has been reported to be responsible from 85% of cases in Buphthalmos. In this study we investigated CYP1B1 gene mutations in the first locus (GLC3A), mapped to chromosome 2p21 in Turkish patients.

DNA samples were isolated from total of 61 individuals (13 familial and 5 isolated cases). CYP1B1 gene was amplified by PCR. Nucleotide sequence of patients who revealed abnormal pattern in SSCP, were screened by DNA Sequence Analysis.

Two different mutations were detected in CYP1B1 gene in buphthalmos patients. The mutations are; 3987 G→A (G61→E) in exon 2 and 8242 C→T (R469→W) in exon 3. The frequency of these mutations in Turkish patients are % 4.5 and % 9 respectively. We also detected five different polimorphisms (3947 cgg/ggg R48→G; 4160 gcc/tcc A119→S; 8125 gcc/gtc A330→V; 8131 gtg/ctg V432→L; 8195 aac/agc N453→S; 8184 gat/gac silent 449) in screened individuals. The frequency of these polymorphisms are %6.6, %14.8, %24, %9.8 and %24 respectively.

The detection of the mutations in CYP1B1 gene will be helpful in early diagnosis of the disease, further understanding of its genetic base and the role of CYP1B1 gene in development and differentiation.

P130

Lyubomira ATANASOVA, Milena PISSARSKA

The role of cytokinin molecules present in RNA for the regulation of plant metabolism is still hypothetical. Largest variety and abundance of cytokinins occur in tRNA while in rRNA their concentrations are lower. The cytokinins in prokaryotic tRNA were found to be affected by environmental and physiological factors. The response of cytokinins in plant RNAs to changes of such factors is quite obscured yet.

In this study the effect of salt stress on the cytokinins in cell RNAs from maize plants grown on liquid nutrient medium was examined. Total RNA was isolated from apical root and shoot parts; tRNA and rRNA were fractionated from the total RNA by Qiagen-anion-exchange chromatography. Cytokinins in hydrolyzed RNAs were detected by indirect competitive ELISA with polyclonal antibodies against trans-zeatin riboside (ZR) and N⁶-(²-isopentenyl)adenosine (iPA).

The treatment of roots with 100 mM NaCl for three weeks gradually reduced root and shoot growth by a half, and decreased root total RNA content but did not change significantly shoot total RNA content. Salt stress altered the level of RNA-containing compounds that cross-reacted in anti-ZR- and anti-iPA-ELISA. An increase of these cytokinin-like compounds was determined in tRNA from roots and shoots at the second day of the stress. One week after exposure to the stress , their level in both, tRNA and rRNA, was lower compared to that in RNAs from control plants. Three weeks after the stress start, the level of cytokinin-like compounds in both RNAs from stressed roots was comparable to the level of control root RNAs and was much lower in stressed shoot RNAs.

In the discussion a suggestion is made, that the abundance and the nature of the cytokinins present in RNAs act as a regulatory mechanism allowing organisms to adapt to the environmental changes.

*Supported by NFSR, project B-1208/02.

d..Molecular structure and function

l.. Metabolic disorders

P131

PROGESTERONE, ESTROGEN AND TESTOSTERONE HORMONES LEVELS IN RATS EXPOSED TO ELECTROMAGNETIC FIELD TO 50 Hz

The effects of extremely low frequency-electromagnetic fields (ELF-EMF) on the biological functions of living organisms represent an emerging area of interest for human health. It has been thought that ELF-EMF can affect neuroendocrine and immune systems. It has been suggested that increased exposure to EMF may have effects on the reproductive system, but evidence from epidemiological studies is inconclusive.

We determined that progesterone, estrogen and testosterone hormones in rats that have experimentally been exposed to an EMF throughout 100 days in order to analyse whether electromagnetic fields have an effect on body progesterone, estrogen and testosterone hormones levels or not. Our aim is to investigate whether there is relation between these hormones and exposing time to EMF.

In our study, 24 Wistar-Albino type female rats were divided two groups First group (n=12) were exposed to EMF throughout 100 days, the second group (n=12) was control group. The experiment group has been exposed to a 0.9 mT -electromagnetic field in Plexiglas boxes for 100 days, 3 hours a day. This field have been prepared with Helmholtz Bobbins. The control group have been kept in Plexiglas boxes under same conditions for 100 days. However, they have not been exposed to a magnetic field. The rats have been sacrification after these exposing periods and progesterone, estrogen and testosterone hormones have been determined in their serum samples. For statistically comparison assessed with Student’s t test using SPSS 10.0.

Testosterone have clearly increased in the rats that have been exposed to the EMF on 100 days (p<0.001). But serum estrogen and progesterone levels did not significantly change.

In conclusion; our result indicated that long-term exposure to ELF-EMF may affect the reproductive activity. ELF-EMF may impair endocrine homeostasis and it may cause peripheral effects.

P132

Işık TÜRKALP¹, Lale UÇAKTÜRK², Hilal SEKBAN³

In this survey in order to search the relationship between the tolerance of glucose and microalbuminuria we analyzed microalbuminuria levels in the first urine sample in the morning, HbA₁C levels in fasting blood and fructosamine levels in serum in 70 cases whom wanted Oral Glucose Tolerance Test (OGTT). For OGTT we analyzed the glucose levels at the 1/2th 1^st, 2^nd and 3^rd hours after giving the cases 75 gr glucose. We evaluated the results according to the criteria of World Health Organization. In 40 cases whose OGTT results were normal we found microalbuminuria level 0.76±0.39 mg/dl, HbA₁C level 4.02±0.32% and fructosamine level 186.7±18.9 µmol/L. In 10 cases whose OGTT results were impared we found the mean microalbuminuria level 1.54±0.77 mg/dl, HbA₁C 5.27±0.53% and fructosamine 223±28.4 µmol/L. In 20 cases whose OGTT results were diabetic we found the mean microalbuminuria level 2.33±1.006 mg/dl, HbA₁C level 6.23±0.57% and fructosamine level 233.6±32.8 µmol/L. According to the OGTT microalbuminuria levels of the diabetic cases (P<0.03) and microalbuminuria levels of the impaired OGTT cases (P<0.05) were considerably higher than the control group. Besides there was a significant difference between the diabetic group and impaired OGTT group (P<0.05). In our research in the term that diabetes hasn’t diagnosed yet the microalbuminuria levels of diabetes and impaired OGTT cases were found higher than control groups. According to these findings it was thought that the microalbuminuria assays that show diabetic nephropathy which was one of the most serious complications of DM would be useful to monitor the disease from the time that DM was diagnosed.

COMPARISION OF ION-SELECTIVE ELECTRODES WITH FLAME EMISION SPECTROPHOTOMETRY AND ENZYMATIC SPECTROPHOTOMETRIC METHOD FOR DETERMINATION OF SODIUM AND POTASIUM IN ABNORMAL SERUM SAMPLES WITH ENDOGENOUS INTERFERENCE

Işık TÜRKALP¹, Ünsal GÜNDOĞDU², Asuman KAPTANAĞASI ORÇUN<sup>3

1</sup>Diamed Dialysis Center, Biochemistry Laboratory, Şişli, İstanbul/TURKEY
²Urfa State Hospital, Biochemistry Laboratory, Şanlıurfa/TURKEY
³Kartal Education and Research Hospital, Biochemistry Laboratory, Cevizli, İstanbul/TURKEY
iturkalp@yahoo.com

In this work to investigate the effects of endogenous interferences to serum electrolyte determinations we have determined sodium (Na⁺) and potasyum (K⁺) concentration in normal 52 healty control serums and 110 abnormal serums with endogenous interference (lipemic, ichteric, uremic and hemolized) by methods ion-selective electrodes (ISE) flame emision spectrofhotometry (FES), and enzimatic spectrofhotometry (ES). In addition accuracy and precision of all these three methods were tested during 14 days with 10 different samples and the correlation with three methods was evaluated with corelation analysis. Effect of endoğgenous interference is observed in hemolized and ichteric serum samples analyses with ES method. Difference between normal control samples and hemolized and ichteric K⁺ samples is very significant (P<0.0001). Except ichteric K⁺ results, FES method is the least affected method from endogenous interference. Moreover ISE method is affected moderately from it. It is observed that ISE results are in excellent agreement with ES results (r_Na = 0,99, r_K = 0,93). ISE and FES results were in agreement, except K⁺ results (r_Na = 0,97, r_K = 0,72). Also ES and FES results were found to be in agreement with each other (r_Na = 0,95, r_K = 0,95). FES and ISE analytical precisions fulfill the target CV_anal < 0,5 Cv_bio. However ES results are found to be appropriate for K⁺ analyses but unacceptable for Na⁺ analyses. As a result it has been found that ES determination method is the most affected method from endogenous interferences whereas ISE method affected moderately and FES method affected the least.

Hülya Ağırdemir¹, A. Akın Denizci², Dilek Kazan^2,3, Altan Erarslan^2,4